Beta-adrenergic receptor (bAR) stimulation serves as the most powerful means to regulate energy metabolism and cardiac performance. Both b1AR and b2AR, the closely related bAR subtypes, are expressed in mammalian hearts, but they display subtype-specific G protein coupling and functions. While b1AR couples to Gs, b2AR couples dually to the Gs and pertussis toxin (PTX)-sensitive Gi proteins with the Gi coupling negating the Gs-mediated contractile response and promoting cell survival in the heart. It has been shown that phosphorylation of b2AR by PKA promotes the receptor-Gi coupling. However, the mechanism inhibiting the b2AR-Gi coupling is poorly understood. Here, we define regulator of G protein signaling 2 (RGS2) as a novel negative regulator of the b2AR-Gi coupling. First, while b1AR-induced contractile response remained intact, b2AR contractile response was profoundly sensitized and resistant to PTX in adult mouse cardiomyocytes cultured for 24 h, in the absence of alterations in the expression of G proteins or the density of either bAR subtype, indicating the b2AR-Gi coupling is severely impaired in cultured cells. Second, RGS2 protein was selectively elevated in cultured cells without changes in other major cardiac RGS proteins, including RGS3, RGS4, RGS5, RGS10 and GAIP. Third, both upregulation of RGS2 and b2AR-Gi uncoupling were fully prevented by bAR stimulation with isoproterenol (1.0 nM) during cell culture, suggesting that the b2AR-Gi coupling is mediated by the receptor activation-induced downregulation of RGS2. This conclusion is corroborated by the fact that gene-targeted knockout of RGS2 sustained the b2AR-Gi coupling in cultured cardiomyocytes even in the absence of bAR agonist stimulation. In sharp contrast, RGS2 adenoviral overexpression abrogated the agonist-induced b2AR-Gi coupling in cultured cardiomyocytes. Thus, RGS2 functions as a powerful negative regulator of the b2AR-coupled Gi signaling, sensitizing b2AR-mediated contractile response in mammalian hearts.